The synaptic vesicle cycle ensures the steady release of neurotransmitters at the nerve terminals from the presynaptic membrane through a sequence of steps from calcium-triggered vesicle fusion at active zones and release of neurotransmitters to the recycling and re-loading of the synaptic vesicles. The recycling of the synaptic vesicles is essential in order to replenish the neuron with new fusion-competent vesicles for subsequent rounds of neurotransmitter release. Various modes of retrieval of synaptic vesicles from the plasma membrane after synapse stimulation have been described, including clathrin-mediated endocytosis. Understanding the molecular mechanisms regulating synaptic vesicle trafficking will provide insight into synaptic function and why-certain neurological diseases arise when these mechanisms are not properly in place. Proteins involved in the synaptic vesicle cycle have been well described and recent identification of proteins involved in lipid metabolism has revealed a role for lipids and their metabolism in the synaptic vesicle cycle. In particular, studies have implicated a fundamental role of phosphatidylinositol-4,5-bisphosphate (PIP2)and other phosphoinositides in this process. Disruption of two main enzymes regulating the levels of PIP2 at the synapse, phosphatidylinositol phosphate kinase type 1 gamma and the phosphoinositide phosphatase synaptojanin 1 (Synjl), was shown to produce defects at multiple stages in the synaptic vesicle cycle, thereby reflecting the multifaceted role of PIP2 in signaling at the synapse. The phospholipid PIP2 is found to be enriched on the plasma membrane and at significantly lower levels in intracellular membranes. This proposal strives to study the mechanisms controlling the spatial restriction of PIP2 dephosphorylation at the plasma membrane that mediate endocytosis of synaptic vesicles. The hypothesis is that the PIP2 hydrolysis machinery utilizes membrane curvature generators/sensors, such as BAR proteins, to eliminate this lipid preferentially from curved membranes rather than flat ones. This idea suggests that PIP2 hydrolysis occurs on endocytic buds rather than the relatively flat plasma membrane. The tight interaction between a membrane curvature generator/sensor, endophilin, and Synj1 has been demonstrated. Our lab has preliminary biochemical data that suggests that PIP2 hydrolysis is affected by membrane curvature. Elucidation of the involvement of the PIP2 in the synaptic vesicle cycle and the mechanisms that control the levels of PIP2 will aid in the further understanding of synaptic function. Defects in the mechanisms that regulate PIP2 lead to impairment of synaptic function and can give rise to neurological disorders.